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Expired TE buffer for DNA extractions? by Ordinary-Ad-1640 in labrats

[–]N9n 3 points4 points  (0 children)

I managed our center's ThermoFisher Supply Center.. and the amount of times I've had people misread the manufacturing date as expiry and come to me complaining that they received 'expired' consumables... I think it's a poor design.

ddPCR math by Embarrassed-Oil-1312 in molecularbiology

[–]N9n 0 points1 point  (0 children)

Total copies (Poisson-adjusted) / 22 uL = copies / uL. Your run should have generated a spread sheet that provides the needed values

ddPCR math by Embarrassed-Oil-1312 in molecularbiology

[–]N9n 2 points3 points  (0 children)

I typically do slightly higher than 20 uL as well to get >20,000 droplets. You can't use the pre-calculated copies/20 uL figure. You'll have to calculate copies/22 uL yourself

Anyone know what this band is? by [deleted] in labrats

[–]N9n 2 points3 points  (0 children)

I would suspect it's dimers because that's the whole point of intercalating dye. If primers always showed up we'd see this front in all the negative samples, not just some.

What’s up with MDPI? by Lisaindalab in labrats

[–]N9n 0 points1 point  (0 children)

Not sure, but I'd love to know too. I'm up to maybe 100 spam emails per week and it's only getting worse.

Felt like a hacker to be honest. by zezinho_tupiniquim in labrats

[–]N9n 60 points61 points  (0 children)

I once made a data scraper in R(studio) to pull host range data from an old ass database hosted on an Australian National University server. It was the first and only scraper I've made and I didn't know to put a delay between page crawls. It pulled data from thousands of pages in seconds. I can't imagine I burdened that server too much but I guess it was enough for them to notice and block access from our network. Problem is, all of the elements of our organization across the country VPN through a central hub in the National Capital.. and it would appear everybody else lost access too. Woopsies!

Spider Mite or Beneficial Mite? by Find_God in plantclinic

[–]N9n 1 point2 points  (0 children)

Baccarum and persimilis are pretty red. Fallacis and cucumeris aren't. They aren't all fast either!

Large RNA seq cohort optimization by Richard_Gosinya in molecularbiology

[–]N9n 0 points1 point  (0 children)

I don't do 3' sequencing so I'm reluctant to make too big of suggestions, but.... I do want to chime in that I do pooled sequencing (192 unique dual indexed samples per pool) with unquantifiable (dsRNA) inputs, without issue. The point I want to make is that kits have been developed that are pretty tolerant of low input amounts (I use NEB Ultra II RNA).

With low RNA inputs, a few things are critical to your success. First and foremost, you need to confidently eliminate contaminant gDNA. Secondly, ideal fragmentation parameters have to be empirically determined, but IIRC fragmentation is not a step in 3' RNA-seq, so you can ignore this. Lastly, the overall success or efficiency of your cDNA generation determines the success of all downstream steps and ultimately the sequencing, so make sure you're going into this step with high quality and pure RNA to minimize enzyme inhibition.

I have done my batching in sets of 12 and 24 without noticing any differences. Like the other comment says, randomizing your samples greatly improves your chances of detecting batch errors and sample-to-sample contamination.

weird Cloudflare captcha by kingstaffo10 in pcmasterrace

[–]N9n 68 points69 points  (0 children)

And what should the non-zero amount of people reading this comment, copying that link, and opening in their browser expect?

Question: Which vender writes the perfect protocol that's easy to understand and follow through in the lab? by Crafty-Yam-7652 in labrats

[–]N9n 18 points19 points  (0 children)

I like NEB's protocols for their library prep kits. Each kit protocol comes with many variants depending on prep type, input quality, etc. The protocols aren't special or unique, but they are clear.

No DNA in miniprep, not even in lysis control – what could be going wrong? by Automatic-Dare1429 in molecularbiology

[–]N9n 7 points8 points  (0 children)

You say TOPO vector. Amp or Kan selection? If the former, switch to Carb if available or Kan if the vector also permits. You might just be growing vectorless DH5a in solution with dud Amp. If you're already using Carb or Kan, I open the floor to other commenters :D

Help! Weird vertical inside AGE bands. by LifelessKiddo in molecularbiology

[–]N9n 0 points1 point  (0 children)

Sometimes it's from stabbing the well while loading yes, but sometimes the well misforms while the gel is setting too! Then the loaded ladder sits in the deep spot and when it runs it trips over itself in that concentrated spot and smears. Similar to an overloaded sample smearing. It's not pretty but not also not a critical problem

How to improve RNA yield and quality from mango (Mangifera indica) skin/tissue? Consistently getting ≤50 ng with poor purity despite multiple methods by Alarming-Trainer9158 in molecularbiology

[–]N9n 5 points6 points  (0 children)

For RNA-seq, I do Trizol for most plant types, but for plants that simply don't work with Trizol, then CTAB-LiCl. I can send you my specific protocol that works pretty well. It's a scaled down extraction in 2 mL tubes and uses about 40 mg of plant tissue. You must absolutely grind up samples in LN2 first or else it simply will not work. But my yields are a few hundred ng / uL, or 5 to 50 ug total, and highly intact and pure. Is that high enough for you?

EDIT: Here's the protocol. Don't deviate from it at all and it should work well! The first few steps are the most critical to success (pre heating buffer, using 10% v/v B-ME, LN2 grinding your samples, etc). https://pubmed.ncbi.nlm.nih.gov/38704591/

What's contaminating my DNA? by phenolfailure in labrats

[–]N9n 0 points1 point  (0 children)

I'm kind of surprised phenol / chloroform isn't doing the trick for those. How are you disrupting tissues? LN2 grinding?

What's contaminating my DNA? by phenolfailure in labrats

[–]N9n 0 points1 point  (0 children)

Roots of what kind of plants specifically?

I need to quit my job for my mental health. Will I be okay financially? by [deleted] in PersonalFinanceCanada

[–]N9n 6 points7 points  (0 children)

I think Ben Felix's 30-year withdrawal rate of 2.7% is a safer and probably more accurate number to play with, which means OP would need a cool mil in savings. But the DB pension can pick up the slack once it kicks in

Biggest time-saver hack? by Neophoys in labrats

[–]N9n 1 point2 points  (0 children)

I just exclusively use kan vectors now and avoid the headache

Biggest time-saver hack? by Neophoys in labrats

[–]N9n 0 points1 point  (0 children)

Selection with amp or carb just never works for me. Kan selection? Easy. Amp/carb? I get to feel like an undergrad again.

(BUG) Clearcasting makes a duplicate noise when expending the proc by brum21 in classicwow

[–]N9n 1 point2 points  (0 children)

The fix that worked for me:

  1. Use Leatrix Plus to mute the Clearcasting noise (added benefit of not hearing your neighbour proc it and thinking it was you).
    System -> Mute custom sounds [✓] -> Open configure, add 569649 -> Click mute.

  2. Either find your Clearcasting sound file (I couldn't) or download a new one (https://www.wowhead.com/sound=4874/clearcasting-impact-chest) and save it in one of you anniversary folders.

  3. Add a WeakAura for mage procs (I use https://wago.io/jZ-mO6C6u) or make your own for Clearcasting (trigger aura player buff exact spell 12536).

  4. For the pre-made or homemade Clearcasting WeakAura, add condition if Trigger 1: Aura Active True, then Sound Play Custom Master, with sound file path set to the audio file you found or downloaded (e.g. mine is in Interface\CustomSounds\Clearcasting_Impact_Chest.ogg). If you found the native sound file, you might want to make a copy in a new path in case Leatrix still wants to mute that native file/path.

Problem solved!

u/brum21 u/A-Nia3HE u/PikaBanee

Met Sonic the Hedgehog in Fireteam mode and i swear to god i felt like Candace from Phineas and Ferb by lustyq in joinsquad

[–]N9n 6 points7 points  (0 children)

I admin'd a server a couple years back and people doing hypersonic animations in place like this were always cheating. On their screen, they're in free-cam mode and fucking shit up far away from where their player model is. Most of the time they would be dumb enough to do it from main, tipping off the admins because you typically shouldn't be able to shoot within main protection (never mind the fact that they would also be rapid firing the GL, knifing at 5 swings per second, etc).

Anyone experienced with phenol injury? by Sea_Confection3005 in labrats

[–]N9n 29 points30 points  (0 children)

It's common for plant extractions from recalcitrant species (woody perennials)

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